Project Summary Between 15 and 20% of couples have difficulty conceiving; failures of the reproductive system thus affect many individuals. In females, understanding the control of ovulation is critical for helping infertility patients conceive single, as opposed to multiple, births while minimizing side effects. The goal of this proposal is to increase our understanding of how the brain responds to ovarian estradiol to generate the central neural signal that ultimately leads to ovulation. This signal is provided by a shift in output of gonadotropin-releasing hormone (GnRH) neurons from one that is strictly episodic, producing on/off GnRH pulses that drive pituitary hormone release, to one in which GnRH release is continuously elevated for several hours. Estradiol initiates this GnRH surge, which induces the luteinizing hormone (LH) surge that triggers ovulation. To induce the GnRH surge, estradiol action switches from negative to positive feedback. Ovariectomized (OVX) mice treated with constant physiological levels of estradiol (OVX+E) undergo daily shifts from negative and positive feedback that are timed to the light-dark cycle, allowing mechanistic studies in a reduced variable model. In ovary-intact mice undergoing reproductive cycles, this switch in estradiol feedback mode occurs on proestrus; our previous work indicates cyclical changes in estradiol induce cycle-dependent changes in the properties of the hypothalamic neurons involved in generating the GnRH surge. This previous work established several mechanisms engaged by estradiol that would lead to suppression of GnRH neurons during negative feedback and activation of these cells during positive feedback. In the proposed work, we will expand upon this base in experiments that range from continued investigation of neurobiological mechanisms to whole animal studies, all aimed at elucidating estradiol feedback and GnRH surge generation. In Aim 1, we will study GnRH neurons as well as kisspeptin neurons in the anteroventral periventricular (AVPV) and arcuate regions, which are postulated to convey estradiol positive and negative feedback to GnRH neurons, respectively. These experiments will move us towards understanding a more complete reproductive neuroendocrine network by studying how synaptic and intrinsic properties interact with one another within these different cell types, and how these cell types interact with each other. We will also study how deleting estrogen receptor (ER) ? in kisspeptin-expressing cells alters the properties of these cells when done prepubertally via cre-lox methods vs in adulthood using CRISPR/Cas9 gene editing. Psychosocial stress interferes with homeostasis and disrupts many physiologic systems including reproduction. In Aim 2, we will study the mechanisms by which acute stress exposure perturbs the shift from estradiol negative to positive feedback, disrupting the LH surge. We will examine how another hypothalamic system, that producing corticotropin-releasing hormone (CRH) interacts with the reproductive neuroendocrine system, examining neurobiological mechanisms by which CRH alters GnRH neuron activity, and the necessity and sufficiency of CRH neurons for stress disruption using genetic approaches.